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Keywords: mycorrhizal inoculants, MYKOVAM®, Narra, nitrogen-fixing bacteria, Pterocarpus indicus

Tree Legume – Microbial Symbiosis and Other Soil Amendments as Rehabilitation Strategies

in Mine Tailings in the Philippines

1National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines Los Baños (UPLB), College, Laguna 4031 Philippines

2Institute of Biological Sciences, College of Arts and Sciences, UPLB, College, Laguna 4031 Philippines

*Corresponding Author: [email protected] [email protected]

Nelly S. Aggangan1*, Julieta A. Anarna1, and Nina M. Cadiz2

A field experiment was conducted to develop rehabilitation protocols for rehabilitating mine tailings areas using arbuscular mycorrhizal fungi (AMFs) and nitrogen-fixing bacteria (NFBs) as microbial biofertilizers. Narra (Pterocarpus indicus) seedlings were inoculated during pricking with AMF with or without NFB Azospirillum spp. After four months in the nursery, the seedlings were planted in a barren, mined-out area in Barangay Capayang, Mogpog, Marinduque, Philippines. During field planting of narra seedlings, vermicompost and lime were mixed with the excavated soil prior to back-filling the 30 cm3 planting hole. Uninoculated narra seedlings were also planted without any amendments, thus serving as the negative control. After one year, 96% (control) and 99% (AMF±NFB) seedling survival were observed with amendments as compared to only 50% in the negative control. The negative control had height and stem diameter of 2.2x and 1.65x, respectively – lower than the control with no biofertilizer but with soil amendments. With soil amendments, mycorrhizal seedlings gave height increases ranging from 98 to 139% and stem diameter from 67 to 87% relative to the uninoculated plants. Mycorrhizal inoculation gave the highest (418 cm3) wood volume while the lowest was the control (50 cm3). The results clearly showed the beneficial effects of microbial biofertilizers and soil amendments for the rehabilitation of mined-out area in Mogpog and could have potential applications for rehabilitation of other mined out areas with similar conditions.

Philippine Journal of Science148 (3): 481-491, September 2019ISSN 0031 - 7683Date Received: 22 Feb 2019

INTRODUCTIONMining is of economic benefit in every country, but there are environmental concerns as large volume (in millions m3) of waste materials are produced every year. The production of large-scale mining wastes has a serious influence on the environment. Mine tailings are the materials remaining after extraction of ores in the mining industry (Gu 2017).

Mine tailings usually contain high concentrations of trace metals and metalloids that can damage land and vegetation (Mendez and Maier 2008). The erosion of the spoil heaps by wind or water and contaminants leaching into groundwater results in permanent pollution of surrounding ecosystems and creates health hazards for local populations (Verdugo et al. 2011).

The Philippines has several abandoned mine tailing areas (Table 1) that need immediate rehabilitation, among which are the mine tailings of Consolidated Mines, Inc.

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(CMI) in Mogpog, Marinduque. The mine tailing area is surrounded by many human settlements and different ecosystems such as marine and agricultural lands, but the most important ones are elementary and secondary schools built just below the mine tailing area. The area has been barren for more than three decades; therefore, surface dust containing heavy metals serves as an aerial pollutant that people can breathe in. The leachates may go into the agricultural areas and marine that could contaminate the local food and water sources. Heavy metals are a health hazard to plants and animals, including humans. In order to reduce the risk, vegetative cover should be established to minimize the health risk. However, establishing

vegetation in mine tailings is often difficult because of their chemical and physical limitations such as metal toxicity, low nutrient content, poor physical soil structure, and low density and diversity of microbial communities (Fernández et al. 2012).

Rehabilitation of mined out areas have used fast-growing tree species such as Eucalyptus and Acacias (Ranjan et al. 2016). Rehabilitation success is achieved with inoculation with beneficial microbes such as mycorrhizal fungi and nitrogen (N) fixers like Azotobacter and rhizobia coupled with suitable soil amendments like compost, farmyard manure, and topsoil (Mazumdar and Kulkarni 2016).

Table 1. List of abandoned mines in the Philippines.

Abandoned mines Location Mineral commodity

1. Benguet Corporation (formerly Benguet Consolidated, Incorporated)a Balabac Island, Palawan Iron, etc.

2. Philex Mining Corporation (Sto. Nino Mines formerly Baguio Gold Mining Company)a

Tublay, Benguet Copper, gold

3. Unidos Mining Corporationa Nabas, Aklan Silica

4. Western Minolco Mining Companya Kapangan, Baguio Copper

5. Zambales Base Metals, Incorporateda Upper Baluno, Zamboanga City Copper

6. Barlo Minesac Mabini, Pagasinan Copper

7. Black Mountain, Incorporatedac Tuba, Benguet Gold

8. Benguet Exploration, Incorporatedac Tuba, Benguet Gold

9. Palawan Quicksilver Mines, Incorporatedac Puerto Princesa City, Palawan Mercury

10. Romblon Marble Minesac Romblon, Romblon Marble

11. Silica Sand Minesac Roxas, Palawan Silica

12. Acoje Mining Company, Incorporated (AMCI)b Sta. Cruz, Zambales Metallurgical chromite

13. Batong Buhay Golds Mines, Incorporatedb Pasil, Kalinga Gold

14. Benguet Corporation – Antamok Mines (BC-BAGO)b Itogon, Benguet Gold

15. Benguet Consolidated Mining Corporation (BCMC)b Itogon, Benguet Gold

16.Benguet Corporation –Dizon Copper/Gold Mines (BCD)b San Marcelino, Zambales Copper

17.Masinloc Chromite Mines –Benguet Corporation (MCM-BC)b Masinloc, Zambales Refractory chromite

18. Filminera Resources Corporation (FRC)b Rosario and Bunawan, Agusan del Sur Gold

19. CDCP-Basay Mineb Basay, Negros Copper

20. Ino and Capayang Mines (ICM)b Mogpog, Marinduque Copper

21. Heritage Mining Corporation Alamag Processing Groupb Llorente, Eastern Samar Chemical chromite

22. Hixbar Gold Miningb Rapu-Rapu Island, Albay Gold

23. Philippine Iron Minesb Jose Panganiban, Camarines Sur Iron

24. Philippine Pyrite Corporationb Bagacay, Samar Pyrite

25. Surigao Consolidated Mining Corporation, Incorporatedb Siana and Mapawa, Surigao del Norte Gold

26. United Paragon Mining Corporationb Paracale, Camarines Norte Gold

27. Vulcan Mining Corporationb Cordon, Isabela Gold

Sources: aMines and Geoscience Bureau, DENR bTetra Tech EM Inc. cMines listed in both sources

Philippine Journal of ScienceVol. 148 No. 3, September 2019

Aggangan et al.: Rehabilitation Strategies in Mine Tailings

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In the Philippines, mined-out and mine tailing areas are commonly devoid of plants presumably due to the toxic effects of residual heavy metals and the infertile state of the soil. Few plants such as ferns and grasses survived in such areas (especially in the gullies) due to their associated microbes.

Mycorrhizal fungi and plants indigenous in mine tailing areas can be tapped for successful rehabilitation of such areas (Reyes et al. 2006, Aggangan and Aggangan 2012). For example, the use of mycorrhizal inoculation in Eucalyptus has been found to increase plant tolerance in copper (Cu) contaminated soil (Aggangan and Aggangan 2006, 2012). Mycorrhizal inoculants and other biofertilizers have been developed and produced in the Philippines at BIOTECH, UPLB, College, Laguna, Philippines. Mycorrhizal inoculants include MYKORICH®, MYKOVAM®, and MYKOCAP® – each containing different species collected from stressed environment such as abandoned mine sites, mine tailings, and marginally acidic, nutrient-deficient areas. The eight (now 12 species since January 2018) species of mycorrhizal inoculant belong to the genera Glomus, Gigaspora, Acaulospora, and Entrophospora. BioNTM , on the other hand, is another BIOTECH-UPLB-developed biofertilizer that contains NFBs identified as Azospirillum spp. The NFB’s were isolated from the roots of Saccharum spontaneum growing in a grassland area in Carranglan, Nueva Ecija in the northern part of Luzon island, Philippines. These biofertilizers are effective in promoting the growth of selected agricultural and forest crops.

MYKOVAM® has been tested on Jatropha curcas as an alternative to biodiesel in the mine tailing of Mogpog (Aggangan et al. 2017). From this study, it was shown that compost improved biomass production – heavier than with lime or MYKOVAM® alone. Better growth was obtained when mycorrhizal inoculation was combined with compost and lime. Compost provides nutrients to the plants and their organic matter (OM) components also have metal-chelating properties that can bind heavy metals in the soil, making them less bioavailable (Ross 1994).

Narra (Pterocarpus indicus, Family Leguminosae) is a high-quality, wood-yielding, fast-growing tree species for walling, furniture making, and the like. It is one of the indigenous premium [i.e., cannot be cut without permission from the Department of Environment and Natural Resources (DENR)] forest species and is the national tree of the country. As a legume, it can form an association with AMFs and NFBs. A nursery experiment conducted by Castillo (1993) reported that dual inoculation using an arbuscular mycorrhizal fungus Glomus macrocarpum + Rhizobium was best for narra grown in Macolod soil. Dual inoculation promoted heavier nodulation, greater sporulation, and root infection

– which in turn increased the yield of dry matter, shoot weight more than root weight, height growth, and uptake of nutrients. Glomus macrocarpum is one of the AMF species in the mycorrhizal inoculant MYKOVAM® biofertilizer. In our study, we report that combined Bio-N

TM and MYKOVAM® has shown a synergistic beneficial effect on narra planted in a red soil marginal grassland in Cavinti, Laguna (Aggangan & Anarna 2013). There are other studies conducted using MYKOVAM® on narra grown on nutrient-deficient soil but not on mine tailings.

The objectives of this study were to develop rehabilitation protocol for mined out and mine tailing areas in Barangay Capayang, Mogpog, Marinduque, Philippines using microbial biofertilizers (mycorrhizal inoculants and NFBs) and other soil amendments (lime, vermicompost, and basal inorganic NPK fertilizer); and to select the best microbial biofertilizer and other soil amendments that promote growth and survival of narra seedlings in mine tailing area.

MATERIALS AND METHODS

Description of Study Site The two-hectare study site is geographically (N13029’54” and S121052’12”E) located in Barangay Capayang, Mogpog, Marinduque, Philippines (Fig. 1). It is part of the 32-ha open-pit, mined-out dumpsite that has been unattended since 1996 after extracting Cu from ore, which is the residual heavy metal after gold extraction. The field experiment was established in a plateau-like hill at an elevation of 60 masl overlooking elementary and high schools built at the foot of the dumpsite that is surrounded by various ecosystems (e.g., mangrove, agricultural, river, and marine) and communities at risk in terms of health and safety. The site was predominantly barren with patches of one- to seven-year-old Acacia auriculiformis, Saccharum spontaneum, Pityrogram macalomelanos, Muntingia calabura, and Trema orientalis. Acacia auriculiformis, a legume tree species from Australia, was introduced in the area by the DENR in the province of Marinduque ten years ago; while T. orientalis, a pioneer tree species, could have been introduced by birds that feed on their fruits. Ferns such as P. macalomelanos and M. calabura, on the other hand, grow on valleys of many mined out areas in the country and are considered heavy metals accumulators. These ferns usually appear about ten years after mining. Spore count of mycorrhizal fungi in the rhizosphere of six different species of ferns ranged 780–3,171 spores per 10 g dry soil and root colonization of 83–85%.

The soil is acidic (pH 5.0; 1:1 soil water) with low OM content (0.52%) plus available Bray P (92 ppm) and K



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was raised from highly acidic to near neutral, while the amount of essential nutrients such as N, P, K, Fe, and Zn was improved.

Table 2. Dutch standards for soil contamination assessment in terms of total concentration of heavy metals in soils.

Element Target value(mg kg–1 soil)

Intervention value*(mg kg–1 soil)

Arsenic 29 55

Barium 200 635

Cadmium 0.8 12

Chromium 100 380

Cobalt 20 240

Copper 36 190

Mercury 0.3 10

Lead 85 530

Molybdenum 10 200

Nickel 35 210

Zinc 140 720

Notes:1. *Intervention value indicates serious contamination of soils where remediation

is necessary.2. For heavy metals, the target and intervention values are dependent on the clay/

silt and OM content of the soils. Standard soil values must be modified by the formula: Lb = Is [(A + B% clay/silt + C% OM)] / (A + 25 B + 10 C) where Lb = intervention values for a particular soil

Is = Intervention values for a standard soil (10% OM and 25% clay)

(0.25 ppm). The soil contained Cu, lead (Pb), cadmium (Cd), and zinc (Zn) (data not shown), although only Cu exceeded beyond the maximum allowable limit of 36 mg kg–1 soil. Soil samples were collected three months prior to field planting in June 2016. Analyses were done at the Central Analytical Service Laboratory (CASL) of BIOTECH-UPLB). After 27 mo, the physico-chemical soil analyses are as follows: pH 6.78 ± 0.52, 2.82 ± 0.69% OM, 0.13 ± 0.03% N, 112.03 ± 38.31 ppm P, 0.38 ± 0.16 cmol kg soil–1 K, 15.68 ± 1.5 cmol kg soil–1 CEC, 127.77 ± 57.9 ppm Fe, 13.17 ± 5.67 ppm Zn, 149.2 ± 41.39 ppm Cu, 0.68 ± 0.11 ppm Cd, and 0.95 ± 0.35 ppm Pb. Even after 27 mo, Cu is still the only heavy metal that exceeded the allowable limit (Table 2). On the other hand, pH

Figure 1. Map of the Philippines showing the project site (inset) in the mine tailing area of Consolidated Mines Inc. in Barangay Capayang, Mogpog, Marinduque (A), which is barren for more than 30 years, where the 1.5 ha field experiments (enclosed in a circle) were conducted (B).

Experimental Design The field experiment was established following a randomized complete block design (RCBD) with five replicates. The treatments were three mycorrhizal inoculants (MYKOVAM®, MYKOCAP®, and MYKORICH®) plus control (no microbial inoculation) and with or without NFB (BioN™). Each treatment was planted with 10 seedlings in a row per block. The planting distance is 2 m x 2 m. Uninoculated control narra seedlings were planted adjacent to that microbial inoculated counterpart that served as the negative control. Plantings were done by the members of the Marinduque Council for Environmental Concern (MACEC). MACEC planted 100 uninoculated narra seedlings with the same spacing but without added lime and vermicompost.

Preparation of Biological Materials Mycorrhizal inoculants with trade names MYKOVAM®, MYKOCAP®, and MYKORICH® contain eight species of AMF belonging to the genera Glomus, Gigaspora, Acaulospora, Scutellospora, and Entrophospora collected and isolated from stressed sites such as grasslands and abandoned mine tailing areas. MYKOVAM®, the first



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mycorrhizal inoculant in 1980, is effective in promoting growth and yield of a variety of crops – including agricultural crops, fruit crops, ornamentals, and forest species.

MYKOVAM® is a commercial mycorrhizal inoculant developed at BIOTECH-UPLB containing three arbuscular mycorrhizal fungal species: Gigaspora margarita, Glomus etunicatum, and Glomus macrocarpum. MYKOVAM®

is effective in promoting growth and yield of a variety of crops – including agricultural crops, fruit crops, ornamentals, and forest species. In 2010, five more mycorrhizal species found effective in promoting the growth of a variety of plant species were added into the MYKOVAM®. On the other hand, MYKOCAP® and MYKORICH® contain the same mycorrhizal species in capsule form but differ in carrier composition.

These mycorrhizal inoculants were developed and produced at BIOTECH-UPLB. MYKOVAM® and MYKOCAP® contain similar mycorrhizal species but differ in form. MYKOVAM® comes in powder form while MYKOCAP® is a capsulized MYKOVAM®. MYKORICH® is a soilless mycorrhizal inoculant in capsule form.

Narra seedlings were raised in Gasan, Marinduque, Philippines by a Bureau of Plant Industry accredited nurseryman. This was done for easier transport of seedlings from the nursery into the field site. The seedlings were inoculated with mycorrhizal inoculants: MYKORICH® (two capsules per seedling), MYKOVAM®

(5 g or one teaspoon per seedling), or MYKOCAP® (two capsules per seedling) with or without BioN™ (one g per seedling) into a one-month-old seedlings growing in individual 4” x 8” poly bags filled with garden soil. Seedlings were field planted after four months in the nursery. During field planting, all seedlings were applied with vermicompost and lime mixed with the excavated soil before backfilling the one square foot hole. In the negative control experiment, uninoculated seedlings were planted without any soil amendments.

Application of Soil AmendmentsLime (calcium carbonate) was bought directly from a manufacturer in Bulacan, Philippines while the compost was bought from Kaharian Realty and Farms Inc., Barangay Adya, Lipa City, Batangas, Philippines under the trade name VERMICAST Organic Soil Conditioner under FPA Registration #1-2LF-1248. The guaranteed analyses (as stated in the package) are: 36% OM, 15:1 C: N ratio, 30% moisture content, 6.8 pH, 1.89% N, 2.49% P2O5, 1.4% K2O, 5.09% Ca, 1.71% Mg, 95.46 ppm Cu, 1,233 ppm Mn, 329 ppm Zn, and 26.34 ppm Fe.

Lime and compost were applied at rates of 500 g and 1,000 g plant–1, respectively; mixed with the excavated

soil; and put it back in a 30-cm3 hole during field planting. The levels of lime and compost used were adjusted based on previous experiments conducted by Naupal et al. (2007) and Aggangan et al. (2017). One month after field planting, 500 g compost, 250 g lime, and 10 g commercial inorganic fertilizer (14-14-14 NPK) were also applied to each seedling and placed in separate 10 cm deep holes 15 cm away from the base of the stem.

Parameters GatheredPlant growth and seedling survival. Seedlings of the same height in each treatment were chosen for field planting (during the onset of the rainy season, June 2016). Initial height and stem diameter were measured one month after field planting and the succeeding measurements were done once in a quarter. Height and stem diameter increments were computed (last quarterly measurements minus the initial measurements) and reported in this paper. The number of surviving or dead seedlings was also recorded.

Plant growth and wood volume. Prior to field planting, five seedlings of similar height and vigor in each of the eight treatments were selected for the determination of plant growth and mycorrhizal root colonization. The seedlings were brought to the Mycorrhizal Laboratory of BIOTECH-UPLB. Height and stem diameter were measured and then the shoots were cut one cm above the root collar. The shoots were washed in distilled water and the leaves were detached from the stem. The roots were gently washed with running water. All adhering dirt and soil were removed carefully using fine-tip forceps. The fine roots (diameter less than 0.5 mm) were separated from the coarse roots and fixed in 50% alcohol for the assessment of mycorrhizal infection. Dry weights of leaves, stem, and coarse and fine roots were obtained after 48 h in a drying oven at 65 °C.

Height and stem diameter of field planted seedlings were monitored every quarter. Wood volume one year after field planting was estimated using the formula below:

Wood volume = (stem diameter2 x height x 3.1416) / 12

Seedling survival was monitored quarterly by counting the surviving and dead seedlings. Percent seedling survival was computed.

Assessment of mycorrhizal root colonization spore population. Fine roots (diameter < 0.5 mm) were cleared with 10% (w/v) KOH solution at 90 °C and stained with 0.05% trypan blue (Phillips and Hayman 1970, Brundrett et al. 1996). Stained roots were viewed under a stereomicroscope and mycorrhizal and non-mycorrhizal infected roots were counted using the gridline intersect method (Giovannetti and Mosse 1989). The presence of attached hyphae, mycelia, vesicles, or arbuscules inside the roots was scored



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as mycorrhiza infected roots. Spores in the rhizosphere soil sample were isolated using the wet sieving and decanting technique (Brundrett et al. 1996). Spores were counted in Petri plates with grid lines under a stereomicroscope.

Assessment of microbial population under field conditions. Rhizosphere soil (about 250 g) of narra seedlings uninoculated or inoculated with either MYKORICH® alone or MYKORICH®+BioN™ were collected after one year in the field. Spores of mycorrhizal fungi were separated from the soil samples following the wet sieving and decanting technique (Brundrett et al. 1996). The population count, based on colony forming units (CFUs), of NFBs was determined using an N-free malate medium (Döbereiner et al. 1976) with modifications. The medium was composed of 5.0 g malic acid, 4.0 g KOH, 0.1 g CaCl2, 0.1 g MgSO4–∙7H2O, 0.1 g MnSO4–∙H2O, 0.9 g K2HPO4, 10 mg FeSO4–∙7H2O, 5.0 mg Na2MoO4– ∙2H2O, 3.0 ml Bromothymol blue 0.5% alcohol solution, and 15.0 g agar. All components were dissolved in 1 L distilled water and the pH was adjusted to 7.0 using 1 M NaOH.

Potato dextrose agar (PDA) was used to screen and estimate the CFUs of fungi in the soil. The media were sterilized at 121 °C at 15 psi. Before pouring the N-free malate medium on the plate, 1 ml/L (v/v) of antifungal (100,000 units Nystatine®; The ACME Laboratories Ltd., Bangladesh) was added to the cooling medium to eliminate the possible fungal contamination. On the other hand, 1 ml of antibiotic (0.1 g ml–1) was added into the PDA to inhibit the growth of bacteria.

A total of 10 g of soil sample was mixed into 90 ml sterile distilled water and shaken vigorously using a vortex to distribute the soil with bacterial cells. Successive 10-fold dilutions were prepared up to 10–5 by transferring 1 ml of suspension from the first dilution to 9 ml sterile distilled water in screw cap tubes with subsequent mixing using a vortex. One-tenth (0.1) ml from each dilution was plated in duplicates into N-free malate medium and PDA, then spread. Fungi were grown for 24 h while the NFB were grown for 3–5 days at 30 °C.

Nutrient and heavy metal concentration. Ten pieces youngest fully expanded leaf samples were collected from each seedling one year after field planting, wrapped separately in paper towels, and placed inside a brown paper bag. Leaf samples were dried at 70 °C for 48 h, ground, and stored in an oven at 60 °C until analysis. N, P, Zn, Cu, and Pb were analyzed at CASL, BIOTECH-UPLB following the procedure of Murphy and Riley (1962). Five grams of dried ground samples were separately wrapped with aluminum foil and combusted at 900 °C in a furnace – after which Cu, Mn, Zn, and Cu concentrations were measured with an atomic absorption spectrophotometer (AA-6701F; Shimadzu, Tokyo, Japan).

Statistical Analyses All data collected were analyzed using analysis of variance (ANOVA) under RCBD. Treatment means were compared using Tukey’s HSD if ANOVA showed a significant effect at p < 0.05. Statistical analyses were done using MSTATC statistical computer program (MSU 1989). Correlation analyses between mycorrhizal association and wood volume, nutrient concentration, and microbial count were done to determine the relationship between these parameters.

RESULTS AND DISCUSSION

Plant Growth and Root Colonization under Nursery ConditionsIn general, dual inoculation with mycorrhizal inoculants and BioN significantly promoted higher height growth of narra after four months in the nursery or prior to field planting (Fig. 2A). The highest (p < 0.001) height was obtained in plants inoculated with MYKOVAM® +BioN™. On the other hand, the lowest was obtained in the uninoculated control (36.9 cm) and BioN™ (40.5 cm) inoculated plants. Inoculation with combined MYKOVAM® and BioN™ increased height growth by 85% relative to the uninoculated control counterpart. There was no significant difference in the height of plants inoculated with single or combined biofertilizers except those with MYKOVAM® alone. In terms of stem diameter, plants inoculated with any of the mycorrhizal inoculants applied singly or in combination with BioN were comparable from each other (Fig. 2B). The highest (p < 0.01) stem diameter was obtained in MYKOVAM® (39%) and MYKOVAM®+BioN™ (40%) inoculated plants and the lowest was in the control (0.48 cm) and those inoculated with BioN™ (0.54 cm). Mycorrhizal colonization was highest and comparable in plants inoculated with MYKOVAM®, MYKORICH®, and MYKORICH®+BioN™ – ranging from 60 to 83% (Fig. 2C). The uninoculated control and those BioN inoculated plants respectively gave a 20 and 10% root colonization percentage by mycorrhizal fungi.

Plant Survival and Growth under Field ConditionsNarra seedlings grew healthy under field conditions depending on the treatments (Fig. 3). One year after field planting, 10% among the control (no microbial inoculation and with no soil amendments) seedlings showed yellowing of leaves as compared to 2% in the uninoculated control counterpart but applied with vermicompost, lime, and NPK. In general, the uninoculated narra applied with soil amendments gave 96% survival, while 98–100% was obtained irrespective of inoculation treatment. In no microbial and no soil amendments, the survival was 50%



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only. These narra seedlings were planted by MACEC (data not shown). Moreover, all the seedlings planted by MACEC exhibited severe nutrient deficiency symptoms and stunted growth. Seedling height was only 36 cm in MACEC-planted narra after 12 months, as compared with 78 cm in narra with no microbial inoculation but with lime and compost and 10 g NPK chemical fertilizer as basal nutrients (Fig. 4A). Soil amendments increased height by 117% and stem diameter by 65% relative to those obtained in narra planted by MACEC (36 cm height and 0.95 cm stem diameter) (Fig. 4B). Mycorrhizal inoculants with or without BioN™ increased (p < 0.01) stem diameter which were comparable from each other. However, in terms of wood volume, inoculation with MYKORICH®

alone gave the highest (418 cm3), which is 7.4 times that of the control (50 cm3) counterpart (Fig. 4C). BioN inoculation increased wood volume by 90%, while the other inoculation treatments increased wood volume by 3–5 times that of the uninoculated control ones.

Microbial PopulationMycorrhizal spore count was highest (145 spores per 10 g soil sample, p < 0.01) in the rhizosphere of narra seedlings inoculated with MYKORICH followed by those inoculated with MYKORICH®+BioN™ (Fig. 5A). BioN™ inoculated seedlings had 29 spores per 10 g soil sample, which was comparable with the control counterpart.

Colony count of culturable fungal population 12 months after field planting was highest (p < 0.001) in the rhizosphere of MYKORICH® (9.9 CFU x 104 g soil-1)

Figure 2. Height (A), stem diameter (B), and mycorrhizal root colonization (C) of four-month-old Narra seedlings inoculated with mycorrhizal fungi with or without BioN (contains NFBs) prior to field planting in a mine tailing area in Barangay Capayang, Mogpog, Marinduque, Philippines. MVAM = MYKOVAM, MCAP = MYKOCAP, MRICH = MYKORICH; N for A and B = 50, N for C = 5.

Figure 3. General appearance of Narra seedlings: just planted (A), and 6 (B) and 12 (C) mo after planting in a mine tailing area in Barangay Capayang, Mogpog, Marinduque, Philippines.


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inoculated narra seedlings. This was followed by those inoculated with MYKORICH®+BioN™ but there was a significant reduction in the fungal colony count, while the lowest (0.82 CFU x 104 g soil-1) was in the uninoculated counterpart (Fig. 5B). Colony count of NFB was highest in the rhizosphere of BioN™ inoculated seedlings. However, there was no significant effect of the inoculation treatments on the colony count of NFB in the rhizosphere of inoculated and the uninoculated narra (Fig. 5C).

Nutrient and Heavy Metal Content in the LeavesN and P were highest (p < 0.001) in the YFEL of MYKORICH®+BioN™ inoculated plants (Table 3). This inoculation treatment increased N concentration in the YFEL by 9.8% and P by 10.7% relative to the uninoculated control treatment (3.26% N and 3,115 ppm P). Zn concentration was highest (p < 0.001) in MYKOCAP®+BioN™ plants, which was comparable with those inoculated with MYKORICH® alone or with MYKOVAM®+BioN™ (Table 3). Cu was lowest in the leaves of MYKOCAP® and MYKORICH® inoculated

Figure 4. Height (A), stem diameter (B), and computed wood volume (C) of Narra seedlings inoculated with mycorrhizal fungi with or without BioN (Azospirillum spp.) after 12 months in a mine tailing area in Barangay Capayang, Mogpog, Marinduque, Philippines. Bars with the same letters are not significantly different from each other using Tukey’s test at p < 0.05. MACEC data was included for comparison only and were not part of the experiment. MVAM = MYKOVAM, MCAP = MYKOCAP, MRICH = MYKORICH; N = 50.

Figure 5. Spore count of mycorrhizal fungi (A), colony count of culturable fungi (B) and NFBs (C) in the rhizosphere soil of Narra seedlings inoculated with mycorrhizal fungi with or without BioN (Azospirillum spp.) after 12 months in a mine tailing area in Barangay Capayang, Mogpog, Marinduque, Philippines. N=5.



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Table 3. Mineral concentration in the youngest fully expanded leaves of Narra seedlings inoculated with different biofertilizers one year after planting in a mine tailing in Barangay Capayang, Mogpog, Marinduque, Philippines. N = 3.

Treatment N% P ppm Zn ppm Cu ppm Fe ppm Pb ppm

Control 3.26 bc*** 3115 ab*** 20.30 b*** 23.54 a* 65.40 a** 3.93 cd***

BION™ 3.27 bc 2468 e 20.65 b 20.62 ab 54.04 ab 6.22 a

MYKOVAM® 3.19 cd 3473 a 18.92 b 20.98 ab 65.00 a 3.59 cd

MYKOCAP® 3.02 d 2544 de 20.57 b 16.91 b 47.22 bc 4.72 bc

MYKORICH® 3.40 b 2590 cde 24.00 a 16.77 b 53.32 ab 5.50 ab

MYKOVAM® +BION 3.34 bc 2992 bc 24.98 a 21.08 ab 46.38 bc 3.02 d

MYKOCAP+BION 3.19 cd 2914 bcd 26.42 a 24.02 a 48.11 bc 3.96 cd

MYKORICH+BION 3.58 a 3448 a 18.06 b 23.27 a 39.54 c 5.69 ab

*,**,*** = significant at p < 0.05, 0.01, and 0.001, respectively.Treatment means in each column with the same letters are not significantly different from each other using Tukey’s test at p < 0.05.

plants, while Fe concentration was highest in the uninoculated one and those inoculated with MYKOVAM® alone and lowest in MYKORICH®+BioN™ inoculated plants. BioN™ inoculation gave the highest (p < 0.001) Pb concentration, which was significant as compared with the other inoculation treatments except those inoculated with MYKORICH® and MYKORICH®+BioN™.

DISCUSSIONAMFs are soil microbes that establish mutual symbiosis with the majority of higher plants, providing a direct physical link between soil and plant root in almost all habitats and climates (Barea and Jeffries 1995, Chaudhry and Khan 2002). They are essential for the successful survival and growth of plants growing in nutrient- (especially P-) deficient derelict soils (Smith and Read 2008).

The mined out area, where the experiment was established, was barren for many years with previous attempts to rehabilitate the area using different plant species. Despite this, some survived but exhibited stunted growth and extreme yellow or purple leaves – indicating either nutrient deficiency or heavy metal toxicity with very low seedling survival. These initial plantings, done in 2000, had no amendments in the soil to make it favorable for plant growth. In this study, the average seedling survival was 98% one year after field planting with the application of lime, compost, and microbial inoculation. The uninoculated ones gave an average of 96% survival, while 98–100% were obtained from the microbial inoculated counterpart. In no-lime, no-compost, and no-microbial inoculation narra seedlings planted by MACEC, the seedling survival was 50%. Moreover, height and stem

growth were 2.2 and 1.65x, respectively – lower than the control with no biofertilizer but with lime and compost amendments. Mycorrhizal narra seedlings gave height increases ranging from 98 to 139% and stem diameter from 67 to 87% relative to the uninoculated control plants. Inoculation with MYKORICH alone gave the highest (418 cm3) wood volume, which is 7.4x that of the control (50 cm3) counterpart. BioN also increased wood volume by 90%, while the other inoculation treatments increased wood volume by 3–5x that of the uninoculated control ones.

Our results conform to previous studies that incorporating compost and inoculating plants with AMF in the rehabilitation of mine tailings areas is beneficial (Wu et al. 2011), including that of Bahraminia et al. 2015) using vetiver grass. Furthermore, Wu et al. (2011) reported that the addition of refuse compost resulted in biomass that was more than three times higher when compared with the control, and was mainly attributed to an improvement in soil properties and nutrient supply vis-à-vis the untreated control. AMF inoculation also significantly increased the dry matter of vetiver by a rate of 8.1–13.8%. Grasses such as vetiver can be planted as the immediate cover of barren, abandoned, mined-out area, but this must be followed with planting of perennial wood plants that are more deep-rooted and with higher biomass. These plant characteristics can reduce soil erosion and can ensure soil stability. The study of Kang et al. (2016) evaluated the potential of six fast-growing, metal-accumulating woody plants for the phytoremediation of heavy metal contaminated soil in China. They found that Viburnum awabuki, Melia azedarach, Ligutrums lucidum, Firminan simplex, Osmanthus fragrans, and Robina pseudoacacial species were appropriate to Cu, Pb, and Cd multi-metal contaminated areas.



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Narra, a legume and indigenous tree species in the Philippines, has not been evaluated for its tolerance to multi-metal contaminated mine tailing and mined out areas. However, with the good and healthy growth and high seedling survival, this species could be of great potential in the revegetation and ecological restoration of Cu mined-out areas of Mogpog – as long as mycorrhizal fungi are introduced coupled with lime and compost amendment. The combined inoculation of mycorrhizal inoculant MYKORICH® and BioN™ facilitated the absorption of the two main essential nutrient elements N and P, and essential micronutrient Cu, thus implying that narra should be inoculated with both symbionts, – the mycorrhizal fungi and the NFB Azospirillum. Being a leguminous tree, narra is associated with Rhizobium; thus, its NFB-forming root nodules could further promote growth and survival in an infertile and toxic condition as in mined-out areas. In the nursery, Rhizobium was not introduced in the soil, but there were nodules of harvested, four-month-old narra seedlings. When these nodules were cut into half, the content is pinkish in color – indicating efficiency in fixing atmospheric N. So far, very limited literatures are available on Rhizobium associated with narra, especially in mine tailings and mined-out areas.

It should also be noted that AMF, NFB, lime, and compost promoted spore production by mycorrhizal fungi and increased colony counts of culturable fungi and NFB in the rhizosphere of narra growing in the experimental site for one year. This indicates that favorable conditions for their production could hinge on changes in micro and macroclimate conditions, as well as a change in the nutrient status of the soil. Llamado et al. (2013) reported an enhanced rhizosphere bacterial population in an abandoned Cu mined-out area planted with Jatropha interspersed with selected indigenous tree species. All the plants in the collection area of Llamado et al. (2013) (adjacent to the present study) were inoculated with mycorrhizal inoculant MYKOVAM® and applied with lime and compost. The field experiment is still in progress where assessment of the changes in the soil’s physical, chemical, and microbial properties for more than three years is to be evaluated. Moreover, micro and macroclimatic factors will be monitored.

CONCLUSIONThe results clearly show the beneficial effects of microbial biofertilizers and soil amendments for rehabilitation of Cu mined-out area in Mogpog. It is recommended that the same field experiments be replicated in other mined-out areas with similar conditions.

ACKNOWLEDGMENTThis project was funded by the Department of Science and Technology – National Research Council for the Philippines (DOST-NRCP) entitled “Rehabilitation Strategies for Rehabilitation of Abandoned Mine Tailing Area in Mogpog, Marinduque” DOST-NRCP Project No. F-155. Acknowledgment is also due to the local government unit (LGU) of Mogpog, Marinduque headed by the Livelo brothers (Senen and Augusto Leo); LGU and residents of Barangay Capayang, Mogpog, Marinduque; Mogpog Police Force; the constituents of the Pantawid Pamilyang Pilipino Program (4Ps); the DENR; DOST Boac; Marinduque Provincial Government Office; and MACEC for their involvement in project during the establishment and for their continuous protection of the field experiments.

REFERENCESAGGANGAN BJS, AGGANGAN NS. 2006. Copper

tolerance of non-mycorrhizal and mycorrhizal Eucalyptus and Acacia seedlings. African J Biotech 5(2): 133–142.

AGGANGAN NS, AGGANGAN BJS. 2012. Selection of ectomycorrhizal fungi and tree species for rehabilitation of Cu mine tailings in the Philippines. J Environ Sci Manage 15(1): 59–71.

AGGANGAN NS, ANARNA JA. 2013. Response of seven indigenous tree species to mycorrhizal inoculant “MYKOVAM® TM” under nursery and field conditions. Proceedings of the 2nd IUFRO International Symposium on Tropical Forest Ecosystem Science and Management; Universiti Putra Malaysia Bintulu Sarawak Campus. 55p.

AGGANGAN NS, CADIZ NM, LLAMADO AL, RAYMUNDO AK. 2017. Jatropha curcas for bioenergy and rehabilitation in abandoned mined out area in Mogpog, Marinduque, Philippines. Energy Procedia 110: 471–478.

BAHRAMINIA M, ZAREI M, RONAGHI A, GHASEMI-FASAEI R. 2016. Effectiveness of arbuscular mycorrhizal fungi in phytoremediation of lead-contaminated soil by vetiver grass. Int J Phytoremediat 18: 730–737.

BAREA JM, JEFFRIES P. 1995. Arbuscular mycorrhzae in sustainable soil-plant systems. In: Mycorrhiza—Structure, Function, Molecular Biology and Biotechnology. Varma A, Hock B eds. Berlin: Springer. 875p.

BRUNDRETT MC, BOUGHER NDB, GROVE T, MALAJCZUK N. 1996. Working with mycorrhizas



490

in forestry and agriculture. ACIAR Monograph 32. Canberra: Australian Centre for International Agricultural Research. 374p.

CASTILLO ET. 1993. Physiological responses of Narra (Pterocarpus indicus Willd.) seedlings inoculated with VA mycorrhiza and Rhizobium in Macolod soils [Ph.D. thesis]. Los Baños, Philippines: University of the Philippines Los Baños – College of Forestry and Natural Resources.

CHAUDHRY TM, KHAN AG. 2002. Role of symbiotic organisms in sustainable plant growth on heavy metal contaminated industrial sites. In: Biotechnology of Microbes and Sustainable Utilization. Rajak RC ed. Jodhpur, India: Scientific Publishers. p. 270–279.

DÖBEREINER J, MARRIEL IE, NERY M. 1976. Ecological distribution of Spirillum lipoferum Beijerinck. Can J Microbiol 22: 1464-1473.

FERNÁNDEZ D, ROLDÁN A, AZCÓN R, CARAVACA F, BÅÅTH E. 2012. Effects of water stress, organic amendment and mycorrhizal inoculation on soil microbial community structure and activity during the establishment of two heavy metal-tolerant native plant species. Microb Ecol 63(4): 794–803.

GIOVANNETTI M, MOSSE B. 1980. An evaluation of techniques for measuring vesicular-arbuscular infection in roots. New Phytol 84: 489–500.

GONZÁLEZ-CHÁVEZ MCA, CARILLO- GONZÁLEZ RC, GODÍNEZ MIH, LOZANO SE. 2017. Jatropha curcas and assisted phytoremediation of a mine tailing with biochar and mycorrhizal fungus. Int J Phytoremediat 19: 174–182

GU HH, ZHOU Z, GAO YQ, YUAN XT, AI YJ, ZHANG JY, ZUO WZ, TAYLOR AA, NAN SQ, LI FP. 2017. The influence of arbuscular mycorrhizal fungus on phytostablization of lead/zinc tailings using four plant species. Int J Phytoremediat 19: 739–745.

KANG W, BAO J, ZHENG J, XU F, WANG L. 2016. Phytoremediation of heavy metal contaminated soil potential by woody plants on Tonglushan ancient copper spoil heap in China. Int J Phytoremediat 20(1): 1–7.

LLAMADO AL, RAYMUNDO AK, AGGANGAN NS, PAMPOLINA NM, CADIZ NM. 2013. Enhanced rhizosphere bacterial population in an abandoned copper mined-out area planted with Jatropha interpersed with selected indigenous tree species. J Environ Sci Manage 16(2): 56–66.

MAZUMDAR S, KULKARNI K. 2016. Remediation of mined out areas and abandoned mines: A case study on Sesa’s approach. Retrieved from http://mines.gov.in/writeaddata on 01 May 2019.

MENDEZ MO, MAIER RM. 2008. Phytoremediation of mine tailings in temperate and arid environments. Rev Environ Sci Biotechnol. 7: 47–59.

MURPHY J, RILEY JP. 1962. A modified single solutions method for the determination of phosphate in natural waters. Anal Chim Acta 27: 31fight club movie36.

[MSU] Michigan State University. 1989. User’s guide to MSTAT-C: Design, Management and Statistical Research Tool. East Lansing, MI: MSU.

NAUPAL R, AGGANGAN NS, PAMPOLINA NM. 2007. Effects of mycorrhizal inoculation and other amendments to copper-rich Mogpog soil to growth and copper-accumulation of Jatropha curcas L. Mycological Society of the Philippines 9th Annual Scientific Meeting and Symposium; Central Luzon State University, Munoz, Nueva Ecija. 30p.

PHILLIPS JM, HAYMAN DS. 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br Mycol Soc 55: 158–161.

RANJAN V, SEN P, KUMAR D, SINGH B. 2016. Reclamation and rehabilitation of waste dump by eco-restoration techniques at Thakurani iron ore mines in Odisha. Intl J Mining Mineral Eng 7(3): 253–264.

REYES SMM, MERCADO GA, LACDAN NF, AGGANGAN NS. 2006. Growth and heavy metal accumulation of narra (Pterocarpus indicus Wild.) in mine waste soils. Proceedings of the 7th Annual Scientific Meeting and Symposium of the Mycological Society of the Philippines; 21 Apr 2006; ERDB, College, Laguna. 29p.

ROSS SM. 1994. Toxic Metals in Soil-Plant Systems. New York: Wiley & Sons.

SMITH SE, READ D. 2008. Mineral nutrition, toxic element accumulation and water retention of arbuscular mycorrhizal plants. In: Mycorrhizal Symbiosis, 3rd edition. p. 145–187.

VERDUGO C, SÁNCHEZ P, SANTIBÁÑEZ C, URRESTARAZU P, BUSTAMANTE E, SILVA Y, GOURDON D, GINOCCHIO R. 2010. Efficacy of lime, biosolids, and mycorrhiza for the phytostabilization of sulfidic copper tailings in Chile: A greenhouse experiment. Int J Phytoremediat 13: 107–125.

WU SC, WONG CC, SHU WS, KHAN AG, WONG MH. 2011. Mycorrhizo-remediation of lead/zinc mine tailings using vetiver: A field study. Int J Phytoremediat 13: 61–74.



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